Description
Background information
The MACSelect Kk - Transfected Cell Selection Kit contains the pMACS Kk.II vector, a control vector, MACSelect Kk MicroBeads, Anti-H-2Kk-FITC, mouse antibody for detection of cells before enrichment, MACSelect Control FITC antibody for detection of cells after enrichment, and a CD14-FITC antibody for analysis of the control experiment when doing a co-transfection. All components are also available separately. The pMACS Kk.II vector encodes the truncated H-2Kk surface marker with an upstream H-2Kk promotor, and can also be used for the expression of a gene of interest driven by a SV40 promotor. Moreover, the vector can also be used for co-transfection with an expression vector encoding the gene of interest. Transfected cells are magnetically labeled with MACSelect Kk MicroBeads and subsequently separated from non-transfected cells.
Note: The expression of H-2Kk is restricted to some rarely used mouse strains. In order to avoid false positive enrichment, cells from these mouse strains, for example, AKR/J or CBA/J should not be selected using this surface marker. Moreover, the H-2Kk marker should be avoided when MHC class I expression is down-regulated either directly or via reduced expression of β2 microglobulin, for example, in some tumor cell lines such as adenocarcinoma cell lines. In those cases, the alternative MACSelect 4 or MACSelect LNGFR Systems should be used instead of the MACSelect Kk System.
The MACSelect Kk System also provides MACSelect Kk Tag Vector Sets allowing for the expression of the gene of interest with an N- or C-terminal tag, including HA, c-myc, or His epitopes. HA-, c-myc, and His-tag expressing proteins can be isolated using µMACS HA, c-myc, or His Isolation Kits, respectively. The combination of these two product solutions helps to effectively streamline transfection with protein interaction studies.
Detailed procedure
Cells are transfected with the transfection method of choice using either the pMACS Kk.II vector encoding the gene of interest for single vector transfection or for co-transfection with an expression vector encoding the gene of interest. The time of optimal marker expression, which determines the incubation time post transfection, has to be taken into consideration for each individual experiment. The level of marker expression can be determined by staining an aliquot of transfected cells with the Anti-H-2Kk-FITC antibody followed by fluorescence microscopy or flow cytometric analysis. Depending on the cell type, transfection method, and culture conditions cells generally need to be cultured between 6 and 84 hours. After incubation, cells are harvested and magnetically labeled using MACSelect Kk MicroBeads. Cells are loaded onto an MS or LS Column placed in a MiniMACS or MidiMACS Separator. After washing, only the magnetically labeled transfected cells are retained on the column. Magnetic separation can also be carried out on the autoMACS Pro Separator. After enrichment, cells can be analyzed by fluorescence microscopy or flow cytometry using the supplied MACSelect Control FITC antibody.
Applications
The MACSelect Kk System facilitates the enrichment of a homogeneous cell population of transiently transfected cells eliminating the need for setting up stable cell lines. As the MACSelect Kk MicroBeads are non-toxic, the isolated cells can be directly cultured. Stable cell lines can also be rapidly and efficiently established by repeated magnetic enrichment without the need for toxic antibiotic treatment. The system can be used for the enrichment of transiently transfected or transduced primary cells to a purity that enables sensitive downstream analysis. It is suitable for transfected cell selection when a rapid, non-toxic and non-immunogenic method is required.
Downstream applications
After enrichment the transfected cells can be subjected to flow cytometry, fluorescence microscopy, (co-)immunoprecipitation experiments, Western blot analysis, or reporter gene assays in functional gene and drug screening, signal transduction, protein interaction, and RNAi knockdown studies.
Overview
The MACSelect Kk System uses the transiently expressed truncated mouse MHC class I molecule H-2Kk as a surface marker to select transfected cells. It can be used in virtually any mammalian adherent or suspension cell line and primary cells in combination with common transfection methods.
